It is generally believed that the human heart cannot regenerate. Instead, following injury, human hearts scar. This inadequate regenerative response contributes significantly to morbidity and mortality in pediatric and adult populations. In preliminary experiments, we have discovered cardiac regeneration in the teleost fish, Danio rerio (zebrafish). The goal of work proposed here is to define the molecular and cellular mechanisms of cardiac regeneration in zebrafish, with the long-term goal of enhancing cardiac regeneration in humans. Aim 1 is to define and characterize models of cardiac injury and regeneration in zebrafish. We hypothesize that zebrafish respond to varying cardiac injuries by regenerating functionally normal myocardium. In preliminary studies, we have developed a surgical model of cardiac injury. We will analyze the extent of cardiac regeneration over time using histochemical, immunohistochemical and electron microscopic techniques. We will characterize the functional properties of regenerated myocardium using electrocardiography, hemodynamic monitoring, and direct inspection. Finally, we will develop a second model of cardiac injury, a thermal injury model using radiofrequency ablation. Aim 2 is to define cellular mechanisms of cardiac regeneration. We hypothesize that zebrafish hearts regenerate through a process of cardiomyocyte proliferation and migration. We will test this hypothesis using immunohistochemical and in situ hybridization techniques. Aim 3 is to define the molecular signature of cardiac regeneration using transcriptional profiling. We will use microarrays to define and characterize the genetic program of cardiac regeneration. Genes of interest will be characterized further by in situ hybridization. Aim 4 is to define molecular mechanisms of cardiac regeneration. We propose to define the functional importance of specific candidate genes using three strategies, genetics, pharmacoeogy, and siRNA. We will take advantage of existing temperature-sensitive mutants, including mps1, sly1, fra7, frd1, frd3, and frd19. When possible, we will test candidates, like FGF receptor genes, using dominant-negative transgenes and existing pharmacologic inhibitors. We will use siRNA to test key candidates. These studies will begin to define the molecular machinery that controls cardiac regeneration.